Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S930/00—Peptide or protein sequence
  • Y10S930/01—Peptide or protein sequence
  • Y10S930/13—Luteinizing hormone-releasing hormone; related peptides

Definitions

• the present invention relates generally to the field of regulating fertility and parturition. More particularly, it concerns the use of unique non-mammalian peptide hormone analogs of GnRH designed to be useful in fertility regulation, post-coital contraception and as a menses-inducing agent.
• the concentration of immunoreactive GnRH-like material in the placenta and maternal blood has been found to vary with gestational age, following a pattern similar to that of hCG. 8 ' 9 It was also demonstrated that exogenous synthetic mammalian GnRH can stimulate hCG production from human placental explants in vitro 10 11 , and that the GnRH stimulation of hCG release was a receptor mediated event, since it was specific and could be inhibited by a GnRH antagonist, [N-Ac-Pro, 1 D-p-Cl-Phe, 2 D-Nal(2) 3 ' 6 ]- GnRH 12 . In additional to the inhibition of hCG, progesterone production was dramatically suppressed.
• hCG response was related to the gestational age of the placenta. ' 3
• a gestational age-related action of the GnRH antagonist on the release of hCG and steroids was observed. 14 Further studies demonstrated a potent action of GnRH on placental prostanoids, 15 ' 16 again resulting in their inhibition when endogenous chorionic GnRH was the highest.
• the GnRH antagonist also inhibited basal prostaglandin production with greater potency than equimolar concentrations of GnRH, and this action was partially conserved by mammalian GnRH.
• GnRH receptor in the pituitary in the pituitary.
• 90 ' 91 It is known that two populations of placental GnRH receptors exist, one having a Ka of 10 "9 M and the other with a significantly lower affinity of 10 "7 M. In addition, superagonist or antagonist for the pituitary GnRH receptor shows very different affinity for the placental receptor.
• 92,93 Other isomers of GnRH, such as salmon GnRH and Chicken II GnRH, have a much greater affinity for the placental receptor, yet bind with a lesser affinity to the human pituitary receptor. 93 These data demonstrate the existence of a specific placental receptor for GnRH-like molecules, yet the true ligand for this receptor is not known.
• GnRH receptor In amphibians, a Chicken II GnRH receptor as well as a mammalian GnRH receptor has been shown. The specificity and evolutionary aspects of the GnRH receptor has been studied in many species. Mammalian GnRH has been reported to be active in many vertebrate classes. Other GnRHs, such as Chicken II GnRH and salmon GnRH, have reduced affinity for the mammalian pituitary receptor.
• GnRH receptor activity as well as the mRNA for the GnRH receptor, varies throughout gestation in the human placenta. 94 ' 95 The receptor is greatest in early gestation and appears to be down regulated by 12-20 weeks. While the receptor is again detectable in term placentas, 94 the mRNA (using a GnRH decapeptide probe and in situ hybridization methodology) was undetectable at this state of gestation.
• placental GnRH receptor can be up regulated in cell cultures by estradiol supports the hypothesis that this receptor is functional in the regulation of placental homongenesis.
• Another factor that regulates a hormone's activity is its metabolism.
• the enzyme that degrades GnRH differs during pregnancy from the enzyme that degrades GnRH in the pituitary or the blood of non-pregnant individuals.
• the primary enzymatic activity for the degradation of GnRH is chorionic peptidase-1 (C-ase-1), a post-proline peptidase.
• C-ase-1 a glycoprotein with a molecular weight of 60,000.
• C-ase-1 acts as a post-proline peptidase, and is inhibited by bacitracin, para-amino- benzamidine, acetopyruvate and certain cations.
• 100 GnRH is actively degraded by C-ase-1 at neutral pH, having a Km of 10 "8 M 102 .
• C-ase- 1 has been localized by the present inventor in the cytoplasm of the syncytiotrophoblast and syncytial buds. It is secreted into maternal blood, where GnRH is not stable without specific inhibitors of this post-proline peptidase.
• 10j C-ase-1 is present in very high concentrations, and accounts for virtually all GnRH degrading activity in the placenta under physiological conditions.
• GnRH its agonistic analogs or antibodies
• to pregnant baboons and monkeys effects a sharp decrease of CG production and progesterone, which in most cases leads to termination of pregnancy.
• ' ,6'122 Interruption of pregnancy was most consistently observed when these mammalian GnRH analogs were administered following implantation.
• administration of low doses of mammalian GnRH does not significantly change circulating hCG.
• 123 ' 124 this finding was dose and gestational age related 125 ' 126 .
• the present invention in a general and overall sense, relates to novel pharmaceutical preparations that include non-mammalian gonadotropin releasing hormone (GnRH) analogs specifically designed to bind human chorionic GnRH receptor and ovarian GnRH receptors. These analogs are designed to be resistant to degradation by chorionic peptidase 1 (C-ase-1). C-ase-1 has been found to specifically and very actively degrade GnRH in chorionic tissues and maternal blood.
• GnRH gonadotropin releasing hormone
• the non-mammalian GnRH analogs of the present invention may act either as a superagonist at the placental receptor leading to its down regulation, or as a pure antagonist of chorionic GnRH at the GnRH receptor.
• the down-regulation or antagonism of endogenous chorionic GnRH will provide for a reduction in human chorionic gonadotropin (hCG) production. This will also provide a reduction in ovarian and placental steroidogenesis.
• hCG human chorionic gonadotropin
• a direct ovarian luteolytic action may be expected to occur. If trophoblastic and/or ovarian function is jeopardized, premature luteolytic action might occur.
• the analogs of the invention may be further defined as resistant to enzymatic degradation by C-ase-1.
• the agonist and antagonists with the greatest receptor affinity and tissue stability are expected to effectively inhibit hCG and progesterone release from human placenta.
• the non-mammalian GnRH analogs of the invention may be used to inhibit placental production of hCG, and have a direct effect on steroidogenesis at the ovary. This physiological effect of the analogs may thus be used to induce luteolysis and menses- induction.
• the invention provides methods of synthesizing analogs of non- mammalian GnRH having increased activity in the chorionic tissues.
• Methods to inhibit hCG production by placental tissues, that in turn provide a reduction of ovarian and placental steroidogenesis, i.e., luteolysis and menses-induction, are provided in another aspect of the present invention.
• the use of these analogs directly on the ovary is yet another particular embodiment of the invention.
• the analogs of the invention may be used in pharmaceutical preparations as a menses-regulating agent, a contraceptive, or as an abortifacient.
• Non-mammalian chorionic GnRH analogs that are superagonist or antagonists at the trophoblastic/placental level constitute yet other embodiments of the invention. Such a non-mammalian analog would provide for the inhibition of steroidogenesis during pregnancy, acting both as an anti-chorionic and anti-luteal agent by inhibiting steroidogenesis leading to menses induction.
• the chorionic GnRH analogs of the invention thus comprise peptides that are capable of specifically binding the chorionic and/or ovarian GnRH receptors with high affinity, are resistant to degradation by the C- ase-1 and effect either a down-regulation of the chorionic GnRH receptor or act as a true antagonist, inhibiting hCG production and ovarian and placental steroidogenesis or directly inhibiting ovarian steroidogenesis.
• the invention comprises a Salmon or Chicken II GnRH sequence, which both show greater affinity for the placental receptor than mammalian GnRH, that are modified at the C-terminal.
• GnRH analog sequence is substituted at the 6-position with a D-Arg, or other D-amino acid.
• both of these modifications are made to the GnRH analog peptide sequence. These modifications are expected to enhance the binding of the molecule, while at the same time inhibit any of the endopeptidases that are present in blood. These analogs are expected to have increased binding to the placental or ovarian receptor and increased metabolic stability.
• the invention provides non-mammalian GnRH analogs with enhanced activity within the intrauterine tissues, as well as a method for regulating hCG production and thus progesterone production during pregnancy.
• These non- mammalian GnRH analogs may also have a direct action at the ovary. Luteolysis may be affected by a dual mechanism i.e., through inhibition of hCG and thus reduction of ovarian steroidogenesis and/or direct inhibition of ovarian steroidogenesis.
• these analogs will be administered intra-nasally, orally intramuscularly or vaginally.
• Treatment with these analogs may require one to three days of active non-mammalian GnRH analog when used as a post-coital contraceptive.
• the placebo is envisioned to start on the first day of menses and continue for approximately 13 days, then the analog would be given days 13 through 28, or less when menses is induced. This could be repeated monthly.
• Numerous IVF protocols now routinely use mammalian GnRH analogs for ovulation timing and have been shown to be nontoxic, even after weeks of administration.
• Another embodiment of the invention provides non-mammalian GnRH analogs that are resistant to degradation by C-ase-1. This analog will bind the chorionic GnRH receptor or non-mammalian GnRH with high affinity so to displace the endogenous GnRH-like activity and block its action.
• the invention provides more potent non-mammalian GnRH analogs that will specifically bind to the placental and the ovarian GnRH receptor.
• analogs will be provided that is stable in maternal circulation and in the blood of non-pregnant individuals. It is also anticipated that these analogs will be biologically active in chorionic tissues and at the ovary in the regulation of hormonogenesis that will affect the maintenance of pregnancy and/or the receptivity of the uterus for implantation. Due to the specificity of these analogs and their relatively short half-life, the present invention provides non-mammalian GnRH analogs.
• proline-containing peptides compete for C-ase-1 activity, such as angiotensin II, and to a lesser extent, thyrotrophin releasing hormone and reduced oxytocin.
• the existing mammalian GnRH analogs are also proline-containing molecules. Since human pituitary and blood contain an enzymatic activity that degrades GnRH at the 5-6 position, not at the 9 position, 103 the present non-mammalian GnRH analogs have been designed to inhibit the former enzymatic activities, and have substitutions in the 5-6 position of the molecule.
• the present analogs are therefore, resistant to degradation at the pituitary or in the blood of non-pregnant individuals, 105 but not the placenta or in maternal blood. Substitution of the Gly'°-NH 2 with ethylamide, or the even more potent -aza-Gly 10 -NH 2 , inhibits degradation by post-proline peptidase. ' 06 A number of the existing analogs also have an ethylamide substitution of Gly'°-NH 2 .
• the initial findings of inhibition can be explained by recognizing that the decapeptide sequences for mammalian GnRH and chorionic GnRH are not identical. Substantial data exists that the receptor and the chemical nature of chorionic GnRH are not identical to GnRH. Postulating that chorionic GnRH differs from the decapeptide, GnRH, and that there is a placental receptor specific for chorionic GnRH, explains the biphasic response of placental hormones. Mammalian GnRH acts as a partial agonist of chorionic GnRH. When receptors are available, it acts as an agonist of chorionic GnRH.
• GnRH When placental receptors are low or occupied, GnRH competes with the more potent chorionic GnRH resulting in an antagonistic action.
• GnRH-like substances have been found by the present inventor to be decreased at mid-pregnancy in women who later have pre-term labor, and increased in those with post term deliveries. In more recent studies, a GnRH binding substance has been demonstrated in their circulation and in these cases hCG was abnormally reduced and pregnancy loss was observed. Thus, the current studies of GnRH-like substance production during pregnancy indicate that chorionic GnRH is of significance to the maintenance of normal pregnancy.
• the present inventor has found that certain non-mammalian GnRH analogs can act on the chorionic GnRH receptor, and with high affinity binding, affect changes in the intrauterine environment that effect the outcome of pregnancy. This finding is the basis of the invention disclosed herein.
• the present investigator has developed particular (non-mammalian) GnRH analogs that can be used for luteolysis and menstrual induction.
• the ability of specific (non-mammalian) GnRH analogs to interact with the physiologic regulation of hCG, progesterone and prostaglandin during luteal phase of the cycle and early pregnancy, may be used to specifically interrupt luteal function and early pregnancy according to the invention as outlined here.
• Figure 1 Action of Chick Il-ethylamide On Degradation of GnRH By C-ase-1. .GnRH 0.00313 M, o GnRH 0.0625 M, V GnRH 0.0125 M, 0 GnRH 0.0250M
• GnRH was actively degraded by C-ase-1. This activity of C-ase-1 was inhibited by, 9 OH-Pro-GnRH, Lamprey, Chicken I-GnRH, Antide, Chicken II-GnRH and Salmon GnRH with a relative potency of 1.5, 1.5, 0.6, 0.6, 0.2 and 0.2, respectively to that for GnRH. Both Chicken II GnRH- 10 ethylamide and 6 Im-btl-D-His-GnRH 10 ethylamide were essentially inactive, i.e., O.001 inhibitory activity for GnRH.
• GnRH was bound by the placental GnRH receptor with a K d of 10- 6 M.
• Chicken II GnRH was similar to GnRH.
• the K d for 6 Im-btl-D-His-GnRH- 10 ethylamide was half the potency of GnRH , while Buserilin and 6 D-Trp-GnRH- 10 ethylamide were twice as active as GnRH.
• the greatest potency having a K d of 3 non-mammalian, i.e. 33-fold more activity than GnRH .
• GnRH by C-ase-1. • GnRH 0.0500 M, oGnRH 0.0250 M, V GnRH 0.012 M, 0 GnRH 0.0062 M
• chorion when used in this application, including the claims.
• the chorion is described as the highly vascularized outer embryonic membrane that is associated with the allantois in the formation of the placenta.
• the present example outlines how analogs of non-mammalian GnRH with increased activity in chorionic and ovarian tissues are synthesized.
• GnRH analogs are designed for activity at the pituitary GnRH receptor and with extended stability in the circulation of non-pregnant individuals.
• the placental receptor binding activity of the different non-mammalian GnRH analogs of the present invention were compared.
• the human placental GnRH receptor is distinct from that at the pituitary.
• Prior mammalian GnRH analogs have been designed to increase activity at the pituitary GnRH receptor and stability in the circulation of non- pregnant individuals. These analogs do not demonstrate potent binding activity at the placental receptor as they do at the pituitary receptor.
• the non-mammalian GnRH's has been designed to interact with preference at the placental receptor and not the pituitary receptor. They have also been designed to limit degradation by the chorionic enzyme, C-ase- 1 , present in maternal circulation as well as the placenta.
• Placental binding activity of the newly synthesized chorionic GnRH analogs have been compared to that for existing pituitary-active analogs of mammalian GnRH.
• Method and Analysis The newly synthesized non-mammalian GnRH analogs and other commercially available analogs were used in placental receptors binding and enzyme stability study described here. On the basis of these studies, the most receptor potent and most enzyme-stable analogs were chosen for further biopotency studies.
• GnRH receptors were purified from the membrane fractions from placentas. The purification procedure for the placental GnRH receptor was performed using a modification of the method described by Bramley et al.
• the non-mammalian analogs' ability to bind to the placental GnRH receptor was compared to that for synthetic mammalian GnRH, Buserilin and the newly synthesized non-mammalian GnRH analogs.
• the more potent analogs were then studied in homologous receptor assays using newly synthesized non- mammalian GnRH analog as the radioiodinated label. This way, the receptor affinity for that analog could be precisely determined.
• Receptors from three different term placentas were used to study each of these analogs.
• the most potent analogs were used for the C- ase-1 stability studies. These data enabled the inventor to predict the most potent non- mammalian GnRH analog structure for the placental GnRH receptor, and assisted in the design of even more potent analogs for the chorionic GnRH receptor.
• the present example demonstrated the utility of using the present invention in controlling and modulating the activity of the placenta, such as in a placenta of a pregnant mammal.
• Mammalian GnRH and its analogs bind to placental receptors.
• the present non-mammalian analogs had not been examined for placental receptor binding.
• chorionic peptidase- 1 A chorionic peptidase activity that actively degrades GnRH in the placenta, named chorionic peptidase- 1 (C-ase-1), was used. This enzyme acts as a post-proline peptidase, and is present in the placenta and in maternal circulation. In a non-pregnant individual very little post-proline peptidase activity is present in blood. Thus, currently available mammalian GnRH analogs have not been designed to be resistant to degradation by this activity. Non-mammalian GnRH analogs were designed with these specific criteria in mind.
• the newly synthesized non-mammalian GnRH analogs are resistant to enzyme degradation and are potent binders of the placental GnRH receptor. Bio-potency was studied using a placental explant system, and by determining the release of hCG, progesterone and prostanoids.
• hCG is the luteotropin of pregnancy, and known to be critical to the maintenance of the corpus luteum during pregnancy. Thus, it is a primary parameter of interest.
• the production of progesterone by the placenta and the ovary is affected by hCG, as well as being independently regulated by a GnRH-like substance.
• Progesterone is primary to the maintenance of uterine quiescence and thus the maintenance of pregnancy, and therefore is of primary interest to these studies. Also, of interest is the effect of these GnRH analogs on prostaglandin production. Prostaglandins are required for abortifacient activity, and thus, the maintenance or increase in their production may be necessary for the proposed action of the analogs.
• GnRH analogs are also expected to act directly at the corpus luteum to inhibit steroidogenesis. These analogs are also expected to be active at the ovarian level.
• the present example demonstrates the isolation of an enzyme from human placentas, and the action of the enzyme as a post-proline peptidase. It actively degrades peptides, such as gonadotropin releasing hormone (GnRH), thyrotrophin releasing hormone (TRH) and Angiotensin II (AGN-II). These peptides contain a proline residue enzyme, chorionic peptidase-1 (C-ase-1).
• the present example also defines enzyme inhibitors of C-ase-1 action on GnRH, such that it might regulate GnRH concentrations within the intrauterine tissues.
• C-ase-1 enzyme activity studies were done by incubating GnRH with C-ase-1 in the presence of varying concentrations of the non-mammalian GnRH analogs. The reaction was stopped by heating at 85°C for 10 minutes. The remaining GnRH was determined using a specific radioimmunoassay. The formation of product, i.e., the N- terminal nonapeptide of GnRH, was calculated by subtraction and its inverse was plotted versus the inverse of the initial substrate to determine the K s of the reaction.
• the inhibitory activity of Antide, 6 Im-btl-D-His-GnRH- 10 ethylamide, 9 OH-Prl-GnRH, Chicken II GnRH- 10 ethylamide, Chicken II GnRH, Chicken I GnRH, Salmon GnRH and Lamprey GnRH was studied. The relative potency of each analog was compared.
• GnRH was actively degraded by C-ase-1. This activity of C-ase-1 was inhibited by, 9 OH-Pro-GnRH, Lamprey, Chicken I-GnRH, Antide, Chicken II-GnRH and Salmon GnRH with a relative potency of 1.5, 1.5, 0.6, 0.6, 0.2 and 0.2, respectively, compared to that for GnRH.
• Both Chicken II GnRH- 10 ethylamide and 6 Im-btl-D-His-GnRH 10 ethylamide were essentially inactive, i.e., O.001 inhibitory activity for GnRH.
• Chorionic peptidase- 1 which is a post-proline peptidase with high specificity for the degradation of GnRH, can also degrade other GnRH species.
• the synthetic mammalian GnRH analogs such as antide are degraded with reduced activity, while other analogs such as Chicken II GnRH- 10 ethylamide and 6 Im-btl-D-His-GnRH 10 ethylamide are resistant to degradation by this endogenous chorionic enzyme. These analogs will be useful in the regulation of chorionic GnRH activity.
• the human placental GnRH receptor shows different kinetic constants for GnRH compared to that of the pituitary receptor.
• Receptor assays were performed by incubating human term placental GnRH receptors with varying concentrations of GnRH or its analogs in the presence of 125 I- Buserilin. The reaction was stopped and the bound hormone precipitated with polyethylene glycol. Following centrifugation the receptor binding activity was calculated and compared for GnRH, 6 Im-btl-D-His-GnRH 10 ethylamide and 6 D-Trp- GnRH- 10 ethylamide, Chicken II-GnRH and Chicken II GnRH- 10 ethylamide.
• EXAMPLE VII - GnRH And Stability Thereof In The Presence of C-ase-1 GnRH was bound by the placental GnRH receptor with a K d of 10 "6 M. Chicken II GnRH was similar to GnRH. The K d for - 6 Im-btl-D-His-GnRH 10 ethylamide was half the potency of GnRH, while Buserilin and 6 D-Trp-GnRH- 10 ethylamide were twice as active as GnRH. The greatest potency, having a K d of 30 nM, i.e. 33-fold more activity than GnRH .
• GnRH analogs were examined for their stability in the presence of C-ase- 1 and placental homogenates. Using the incubation system developed for the C-ase-1 activity, the degradation of each analog was studied. Previously, this method was used to determine the degradation of GnRH by C-ase-1. Each of these analogs was studied for their ability to act as competitive inhibitors of GnRH for C-ase-1 activity (Table 1). The inverse of the product was plotted against the inverse of the original substrate concentrations to determine Ks of the competition. The K, was determined by plotting the inverse of the product formed verses the inhibitor used.
• the stability of these analogs in the present of whole placental homogenates was examined.
• the ethylamide derivative has a slowed degradation rate as compared to GnRH, but can be degraded.
• Chicken II and its ethylamide analog are more stable than the mammalian GnRH analogs analyzed to date.
• the hCG inhibiting activity of the GnRH analogs was studied using an in vitro human placental explant system.
• the newly synthesized GnRH analogs are resistant to enzyme degradation and one potent binders of the placental receptor.
• the bio-potency was done with a placental explant system, and the release of hCG, progesterone and prostaglandin E 2 was assessed.
• hCG is the luteotropin of pregnancy and know to be important in the maintenance of the corpus luteum during pregnancy.
• the production of progesterone by the placenta is affected by hCG, and may be independently regulated by GnRH as well.
• Progesterone is primary to the maintenance of uterine quiescence and thus the maintenance of pregnancy. Of interest was the effect of these GnRH analogs on prostaglandin production. Prostaglandins are required for abortifacient activity.
• D-Arg(6)-Chicken II GnRH analog-NH 2 has bioactivity in the regulation of hCG and progesterone production in the human term placenta.
• the present example defines a method by which the present invention may be used to maintain pregnancy in a pregnant mammal.
• the mammal in some embodiments is a pregnant human.
• a proposed dose regimen it is anticipated that a pregnant female between 100 lbs and 150 lbs would be administered about 10 nanogram to 1.0 gram of
• the dosing regimen will comprise a pulsatile administration of the Chicken II GnRH over a 24-hour period, wherein the daily dosage is administered in relatively equal l/24 th fractions.
• the daily dosage is about 2.4 micrograms
• the patient would be administered about 0.1 micrograms per hour over a 24-hour period.
• Such a daily pulsatile administration would create a hormonal environment in the patient sufficient to maintain pregnancy.
• the particular pharmaceutical preparations may be created by one of skill in the pharmaceutical arts. Remington's Pharmaceutical Sciences Remington: The Science and Practice of Pharmacy, 19 th edition, Vol. 102, A.R. Gennaro, ed., Mack Publishing co. Easton, PA (1995), is specifically incorporated herein by reference for this purpose.
• the present example demonstrates the utility of the present invention for use as a post-coital contraceptive preparation.
• analogs defined here, and conservative variants thereof maybe formulated into a pharmaceutically acceptable preparation, and then administered to a female mammal having been inseminated during the prior 24 to 72 hours (prior 1 to 3 days). Relatively high doses of about 0.1 gram to about 10 grams of the non- mammalian GnRH analog would be given daily for 2 to 5 days, on the average about 3 days.
• GnRH may be prepared as part of a suitable vector, such as in an adenovirus or retroviral vector, and administered to the animal. Once the sequence is incorporated into the cell, the peptide product will be translated and peptide supplied. Because this method of treatment would not require that the peptide travel in the blood circulation in order to reach the site of action, there would be no requirement that the analog possess enzyme degradation resistance. This mode of treatment has not thus far been proposed, and hence the use of such a method in the regulation of female fertility is a novel clinical regimen.
• the non-mammalian analogs are also contemplated to be useful to directly affect the ovary. By way of example, this technique renders the system useful as a contraceptive.
• the non-mammalian GnRH analog would be given daily from the start of ovulation and continue for 8 days to two weeks, stopping with onset of menses.
• the present example demonstrates the utility for using the present invention non- mammalian GnRH analog decapeptides to prepare antibodies that preferentially bind the GnRH peptide sequences, or that bind the ovarian, placental or any other non-pituitary GnRH peptide or protein. It is anticipated that these non-mammalian GnRH analog antibodies may be used in a variety of screening assays. For example, these antibodies may be used to determine levels of GnRH are present, in a sample as an indicator molecule. The levels of such GnRH may be used to monitor and follow a patient's pregnancy, as well as an indicator of the length of gestation.
• the antibodies to non- mammalian GnRH may be monoclonal or polyclonal antibodies.
• Polyclonal antibodies may be created by standard immunization techniques, wherein the immunogen used will be the non-mammalian Chicken-II GnRH analog or the Salmon GnRH analog decapeptide described herein. These peptides may be used either alone or together in a pharmaceutically acceptable adjuvant.
• the animal, such as a rabbit, would be administered several doses of the decapeptide preparation, and the levels of the animal's antibody blood levels monitored until an acceptable antibody level (titer) had been reached.
• the spleen of the animal would be harvested, and then fused with an immortalized cell line, such as a cancer cell line, to produce a population of hybridoma cells.
• an immortalized cell line such as a cancer cell line
• This hybridoma population of cells would then be screened for those that produce the highest amount of antibody that specifically bind the non-mammalian GnRH analog decapeptide.
• Such hybridoma cells would be selected, and then cultured.
• the antibody to non-mammalian GnRH would then be collected from the media of the cell culture using techniques will known to those of skill in the art.
• Petraglia F, Lim AT, Vale W 1987 Adenosine 3', 5-monophosphate, prostaglandin, and epinephrine stimulate the secretion of immunoreactive gonadotropin-releasing hormone from cultured human placental cells. J Clin Endocrinol Metab 65:1020-1025.
• GnRH gonadotropin-releasing hormone
• Genazzani AR 1994 Pulsatile fluctuations of plasma-gonadotropin-releasing hormone and corticotropin-releasing factor levels in healthy pregnant women. Acta Obstet Gynecol Scand 73:284-289. 111. Seshagiri PB, Terasawa E, Hearn JP 1994 The secretion of gonadotropin- releasing hormone by peri-implantation embryos of the rhesus monkey: comparison with the secretion of chorionic gonadotropin. Hum Reprod 9:1300- 1307.

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